Arthropod-borne flaviviruses cause a wide range of important human diseases for which there are no specific therapies. To address this critical shortfall in preparedness to confront these emerging and re-emerging viruses, the goal of this proposal is to discover novel drug targets and develop new antiviral therapies that can be used broadly to treat a variety of flaviviral infections. A partnership of four independent academic laboratories, two at Duke Univeristy and two at the University of Virginia, will attack crucial aspects of pre- clinical drug development, as organized into the following specific aims: (1) Identification of high-quality targets for pan-flaviviral therapeutics. In this aim, three independent yet complementary approaches will be used to identify cellular targets for broad-spectrum therapies capable of treating a diverse group of flaviviral illnesses. These include genome-scale RNAi screens to identify novel host factors required for flaviviral infection, mining of the human purinome to find purine-binding proteins that support infection, and application of a yeast-based drug discovery platform for the identification of novel targets. (2) Chemical screens for inhibitors of flavivirus replication. Targets identified in Aim 1 will be evaluated, and a subset will subjected to small molecule library screens using proteome mining and yeast-based methodologies. Priority will be given to compounds showing broad-spectrum activity including efficacy against mosquito-borne flaviviruses. (3) SAR and medicinal chemistry to define pan anti-flaviviral inhibitors. Up to six independent medicinal chemistry programs will be carried forward based on the most attractive products of the screens from Aim 2. Lead compounds will be optimized for potency of in vitro binding and efficacy in tissue culture models. (4) Pre-clinical testing. Established mouse models of YFV and WNV infection and newly established models of DENV infection will be used for maximum tolerated dose and initial pharmacological and efficacy studies. Flaviviruses are an emerging threat to public health in the US, a current risk to our armed forces and other citizens deployed around the world, and a major problem globally. At this time there is little that can be done to prevent or treat the majority of flaviviral infections and therefore development of broad-spectrum anti-flaviviral drugs is of crucial importance.